DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR SIMULTANEOUS

DETERMINATION OF HALOPERIDOL AND REDUCED HALOPERIDOL

IN PLASMA: APPLICATION IN PHARMACOKINETIC STUDY

by

THEEPA ASUALINGAM

Thesis submitted in fulfillment of the requirement for the Degree of

Master of Science

April 2007

ii

To my beloved Sidha Yogi Siva Sangkara baba, parents, husband, son,

parents-in-law and sisters.

iii

ACKNOWLEDGEMENTS

First and foremost, I wish to express my utmost gratitude and heartfelt

appreciation to my dedicated principal supervisor, Professor Sharif Mahsufi Mansor for

his guidance, invaluable advice and encouragement throughout my graduate program. I

am most grateful for all the scientific knowledge I have gained from him and all the

fruitful discussions we have had. I would like to thank my co-supervisor, Dr. Surash

Ramanathan for always being so enthusiastic about the projects I have been involved

in and for his endless support throughout my studies.

I am also taking this opportunity in thanking Professor Yuen Kah Hay for

providing his support and lab facility and Dr. Ng Bee Hong for her expert technical

assistance during animal study. Thanks also to the Janssen foundation for providing

reduced haloperidol. I am grateful to the Institute of Graduate Studies, USM for

providing the financial assistance through the Graduate Assistance Scheme during my

candidature.

I would specially like to thank Mr. A.K.M. Mahbubuzzaman for being

with me throughout and sharing my joys and disappointments. Not forgetting also my

dear friends Kanabathy, Shubatra Pillay, Lai Choon Shen, G. Venkatesh, Chitra and

Venessa Daniel. I would also like to acknowledge my gratitude to all the staff and lab

assistants of CDR, especially to Mr R. Arunachalam and Mr. M.Asokan.

My deepest gratitude goes to my parents for their continuous

encouragement, support and interest throughout my studies. Very special thanks to my

husband, Mr. N. Ragu for his patience, constant encouragement, unconditional love as

well as financial and moral support during my research.

iv

TABLE OF CONTENTS

Page

TITLE i

DEDICATION ii

ACKNOWLEDGEMENTS iii

TABLE OF CONTENTS iv

LIST OF TABLES x

LIST OF FIGURES xii

LIST OF SYMBOLS AND ABBREVIATIONS xiv

ABSTRAK xvii

ABSTRACT xix

CHAPTER 1- INTRODUCTION

1.1 Psychosis 1

1.1.1 Anxiety 1

1.1.2 Mania or bipolar disorder 1

1.1.3 Depression 2

1.1.4 Schizophrenia 2

1.2 Antipsychotic drug 3

1.2.1 Typical antipsychotic drug 3

1.2.2 Atypical antipsychotic drug 3

1.3 Haloperidol 4

1.3.1 Assay of HP and RH in biological fluids 7

1.3.2 Pharmacology of HP 9

1.3.3 Pharmacokinetic of HP and RH 16 1.3.4 Drug interactions 23

1.4 Bioanalytical method development 27

v

1.4.1 Introduction to HPLC 27

1.4.2 Column 27

1.4.3 Mobile phase 28

1.4.4 Sample preparation 30

1.5 Bioanalytical method validation 31

1.5.1 System suitability test 31

1.5.1.1 Capacity factor 32

1.5.1.2 Resolution 32

1.5.1.3 Tailing factor 32

1.5.2 Selectivity 32

1.5.3 Recovery 33

1.5.4 Linearity 33

1.5.5 Quality control 34

1.5.6 Precision 35

1.5.7 Accuracy 35

1.5.8 Limit of detection 35

1.5.9 Lower limit of quantitation 36

1.5.10 Stability 36

1.5.10.1 General acceptable criteria for Stability studies 37

1.5.10.2 Stock solution stability study 37

1.5.10.2 On bench stability study 37

1.5.10.3 Freeze thaw stability study 37

1.5.10.4 Processed samples stability study 38

1.5.10.5 Long-term stability study 38

1.6 General principle of pharmacokinetics 39

1.6.1 Drug absorption 39

1.6.2 Drug distribution 41

vi

1.6.3 Drug elimination 43

1.6.4 Drug metabolism 43

1.6.5 Drug excretion 45

1.7 Pharmacokinetic parameters 47

1.7.1 Elimination half-life 48

1.7.2 Clearance 48

1.7.3 Area under the curve 50

1.7.4 Apparent volume of distribution 50

1.7.5 Mean residence time 51

1.7.6 Peak plasma concentration 52

1.7.7 Time to peak concentration 52

1.8 The aim of the thesis 52

CHAPTER 2 - CHEMICALS, INSTRUMENTS AND STANDARDS

2.1 Chemicals 54 2.2 Instruments 54 2.3 Standard solutions 55

2.4 Preparation of extraction solvents and buffer 56

2.5 Standards and purity 57

2.4 Silanisation of glassware 57

CHAPTER 3 - METHODOLOGY

3.1 Analytical method development 61

3.1.1 UV spectrum 61

3.1.2 Column selection 61

3.1.3 Development of mobile phase 61

3.1.4 Detector linearity 62

3.1.5 Internal standard 62

vii

3.1.6 HPLC chromatographic condition 62

3.2 Extraction method for HP and RH in plasma 63

3.2.1 Extraction solvent 63

3.2.2 Basifying agent 63

3.2.3 Rotamix mixing time and speed 63

3.2.4 Back extraction solvent 63

3.2.5 Extraction procedure 64

3.3 Bioanalytical method validation 64

3.3.1 System suitability test 64

3.3.2 Selectivity 65

3.3.3 Recovery 65

3.3.4 Calibration curve 65

3.3.5 Quality control 66

3.3.6 Assay precision and accuracy 66

3.3.7 Lower limit of quantification and limit of detection 66

3.3.8 Stability study 67

3.3.8.1 Stability in solvent 67

3.3.8.1.1 Stability of HP, RH and PYR in methanol 67

3.3.8.1.2 Stability of HP and RH in

reconstitute solvent 68

3.3.8.2 Stability of HP and RH in plasma samples 68

3.3.8.2.1 On bench stability 68

3.3.8.2.2 Freeze-thaw stability 69

3.3.8.2.3 Processed stability 69

3.3.8.2.4 Long-term stability 69

3.4 Cross validation of pharmacokinetic assay method 70

3.5 Pharmacokinetic study in rats 70

3.5.1 Animals and chemicals 70

viii

3.5.2 Study protocol 72

3.6 Data analysis 72

CHAPTER 4 - RESULTS AND DISCUSSION

4.1 Development of HPLC chromatographic condition 74

4.1.1 UV spectrum 74

4.1.2 Reconstitute solvent 75

4.1.3 Column selection 78

4.2 HPLC method development 80

4.2.1 Effect of methanol on capacity factor of HP, RH and PYR 80

4.2.2 Effect of buffer pH on capacity factor of HP, RH and PYR 83

4.2.3 Effect of potassium phosphate buffer concentration on peak asymmetry of HP, RH and PYR 84

4.3 Detector linearity 88 4.4 Internal standard 88

4.5 Extraction method development and optimization 89

4.5.1 Extraction solvent optimization 89

4.5.2 Effect of basifying agent 92

4.5.3 Effect of rotamix mixing time and speed 94

4.5.4 Effect of back extraction solvents 98

4.6 Bioanalytical method validation 101

4.6.1 Selectivity 101

4.6.2 Calibration curve 104

4.6.3 Accuracy and precision 107

4.6.4 Lower limit of quantification and limit of detection 107

4.6.5 Stability in methanol 109

4.6.6 Stability of HP, RH and PYR in reconstitute solvent 115

4.6.7 Plasma samples stability 115

ix

4.6.7.1 On bench study 115

4.6.7.2 Freeze-thaw cycle 118

4.6.7.3 Processed samples 118

4.6.7.4 Long-term stability 121

4.7 Cross validation of pharmacokinetic assay method 126

4.8 Pharmacokinetic study in the rats 127

CHAPTER 5 - SUMMARY AND CONCLUSION 132

CHAPTER 6 – RECOMMENDATION FOR FUTURE STUDY 134

REFERENCES 135

APPENDICES 152

PUBLICATION 157

x

LIST OF TABLES

Page

Table 1.1 Summary of analytical method for HP determination in biological matrix. 11

Table 1.2 Summary of extraction method for HP determination in biological matrix. 14

Table 1.3 Summary of pharmacokinetic for HP and RH. 17

Table 1.4 Concentration of HP and RH in rat tissues after a single intraperitoneally injection of HP or RH. 18

Table 1.5 Concentration of HP and RH in striatum and plasma

after repeated IP injections of HP. 19 Table 1.6 Pharmacokinetic parameters after administration

of HP and RH. 19 Table 1.7 Pharmacokinetic interactions between HP and

concomitant drugs. 26

Table 1.8 Phase I reactions. 46

Table 1.9 Phase II reactions. 46

Table 4.1 System suitability study. 87 Table 4.2 HP recovery percentage from plasma using various

back extraction solvents (mean ± SD, n=5). 99 Table 4.3 RH recovery percentage from plasma using various

back extraction solvents (mean ± SD, n=5). 100 Table 4.4 Retention time of the tested drugs for

selectivity study. 103

Table 4.5 Back calculated values of the calibration samples (n=5) of HP and RH in plasma. 106

Table 4.6 Within-day and day-to-day precision and accuracy

of HP and RH in spiked plasma. 108 Table 4.7 Stock solution stability data of HP in MeOH. 112 Table 4.8 Stock solution stability data of RH in MeOH. 113 Table 4.9 Stock solution stability data of PYR in MeOH. 114 Table 4.10 Stock solution stability data of HP and RH in

reconstitute solvent at room temperature (25 ± 10 C). 116

xi

Table 4.11 On bench (8 h) stability of HP and RH in plasma

at room temperature (25 ± 1°C). 117 Table 4.12 F-T (3) cycles stability data of HP and RH in plasma. 119

Table 4.13 Processed sample stability data of HP and RH in plasma. 120

Table 4.14 Long-term stability data of HP in plasma (-20°C). 122

Table 4.15 Long-term stability data of RH in plasma (-20°C). 123

Table 4.16 Long-term stability data of HP in plasma (-85°C). 124

Table 4.17 Long-term stability data of RH in plasma (-85°C). 125

Table 4.18 Mean plasma concentrations (ng/ml) of HP in rats after administration with 2.5 mg/kg HP orally. 129

Table 4.19 The pharmacokinetic parameters HP after oral

administrations of HP solution 2.5 mg/kg in rats. 130

xii

LIST OF FIGURES

Page

Figure 1.1 Structural formula of typical antipsychotic drugs. 5

Figure 1.2 Structural formula of atypical antipsychotic drugs. 6

Figure 1.3 Metabolism of HP. 22

Figure 2.1 Stock solution dilution sequences for HP and RH. 58

Figure 2.2 Dilution sequence for HP or RH stability samples. 59

Figure 2.3 Dilution sequence for PYR stability samples. 60

Figure 3.1 Preparation of long-term stability samples in plasma. 71

Figure 4.1 UV absorption spectrum of HP (50 µg/ml) in MeOH. 76

Figure 4.2 Chromatogram of standard PYR, RH and HP (A) prepared in MeOH. (B) diluted in mobile phase (50 mM KH2PO4(pH 5.0) – MeOH, 51:49 v/v). (Peaks:1:PYR, 5 ng/ml; 2:RH, 50 ng/ml; 3:HP, 50 ng/ml). 76

Figure 4.3 Chromatogram of standard PYR, RH and HP diluted in reconstitute solvent (50 mM KH2PO4 (pH 5.0) – MeOH, 60:40 v/v). (Peaks:1:PYR, 5 ng/ml; 2:RH, 50 ng/ml; 3:HP, 50 ng/ml). 77

Figure 4.4 A representative chromatogram of standard PYR, RH and HP separated on Merck C18 column. (Peaks:1:PYR, 5 ng/ml; 2:RH, 50 ng/ml; 3:HP, 50 ng/ml). 79

Figure 4.5 A representative chromatogram of standard PYR, RH

and HP separated on Inertsil C8-3 column. (Peaks:1:PYR, 5 ng/ml; 2:RH, 50 ng/ml; 3:HP, 50 ng/ml). 79

Figure 4.6 A representative chromatogram of standard PYR, RH and HP.

Mobile phase: 50 mM KH2PO4 (pH 7.0)- MeOH (50:50,v/v) (Peaks: 1: PYR, 5 ng/ml; 2:RH, 50 ng/ml; 3:HP, 50 ng/ml). 82

Figure 4.7 Influence of MeOH percentage on capacity factor. Mobile phase : 50 mM KH2PO4 (pH 5.0 ) – MeOH.

Key : (ï) PYR; (] ) RH; (X) HP. 82

Figure 4.8 Influence of KH2PO4 buffer pH on capacity factor. Mobile phase composition:

50 mM KH2PO4 - MeOH, (49:51, v/v). Key : (ï ) PYR; (] ) RH; (X) HP. 86

xiii

Figure 4.9 Influence of KH2PO4 buffer concentration on

peak asymmetry. Mobile phase : KH2PO4 ( pH 5.0) - MeOH, ( 49:51, v/v). Key : (ï ) PYR; (] ) RH; (X) HP. 86

Figure 4.10 Detector linearity curves (n=5) for HP( ]) and RH( H)

in reconstitution solvent (50 mM KH2PO4 buffer (pH 5.0) – MeOH, 60:40, v/v). 87

Figure 4.11 Influence of the percentage of isoamylalcohol in hexane

on HP recovery from plasma (mean ± S.E.M., n=5). 91

Figure 4.12 Influence of the percentage of isoamylalcohol in hexane

on RH recovery from plasma (mean ± S.E.M., n=5). 91 Figure 4.13 Influence of borate buffer pH on HP recovery

from plasma (mean ± S.E.M., n=5). 93

Figure 4.14 Influence of borate buffer pH on RH recovery

from plasma (mean ± S.E.M., n=5). 93 Figure 4.15 Influence of mixing time on HP recovery

from plasma (mean ± S.E.M., n=5). 96

Figure 4.16 Influence of mixing time on RH recovery

from plasma (mean ± S.E.M., n=5). 96

Figure 4.17 Influence of mixing speed on HP recovery

from plasma (mean ± S.E.M., n=5). 97 Figure 4.18 Influence of mixing speed on RH recovery

from plasma (mean ± S.E.M., n=5). 97 Figure 4.19 A representative chromatogram of extracted human plasma

spiked with 40 ng/ml of HP and RH and 5 ng/ml PYR (IS). Peaks : 1. PYR, 2. RH, 3. HP. 102

Figure 4.20 A representative chromatogram of extracted drug free plasma. 102

Figure 4.21 Calibration curves (n=5) for HP() and RH(♦) in plasma. 105 Figure 4.22 Chromatogram after extraction of a plasma sample

spiked with 1.0 ng/ml of HP and RH and 5 ng/ml of PYR Peaks : 1. PYR, 2. RH, 3. HP. 110

Figure 4.23 A representative chromatogram of: (A) Extracted drug free rat plasma and (B) extracted 4.0 h plasma samples of rat given 2.5 mg/kg oral dose of HP. Peaks : 1. PYR, 2. HP. 131

Figure 4.24 The mean plasma concentration versus time curve of

HP in rats (n=7) after oral administration of 2.5mg/kg HP. 131

xiv

LIST OF SYMBOLS AND ABBREVIATIONS

ABBREVIATION Full name

ACE Animal Ethics Committee

ACN Acetonitrile

AGP Alpha-1-acid glycoprotein

AHFS American hospital formulary services

As Asymmetry factor

AUC 0-12 Area under the curve from time zero to 12 h.

AUC 0-∞ Area under the curve from time zero to infinity.

AUFS Absorption units full scale

BP British Pharmacopoeia

C18 Column having octadecyl chain of C atom

C8 Column having octadecyl chain of C atom

CL Clearance

Cmax Peak plasma concentration

CV Coefficient of Variation

CYP Cytochrome P450

CYP2D6 Cytochrome P450 subfamily 2D6

CYP3A4 Cytochrome P450 subfamily 3A4

D2 Dopamine subtype receptor 2

et al. Co-workers

FDA Food and Drug Administration

F-T Freeze-thaw

g Gram

h Hour

HP Haloperidol

xv

HPLC High Performance Liquid Chromatography

HPLC-EC High Performance Liquid Chromatography coupled with electrochemical detector

HPLC-UV High Performance Liquid Chromatography coupled with

ultraviolet detector ICH International Conference on Harmonization IS Internal standard

k’ Capacity factor

K+ Potassium ion

KH2PO4 Potassium dihydrogen phosphate

l Liter

LLOQ Lower limit of quantification

LLE Liquid-liquid extraction

LOD Limit of detection

M Molar

MeOH Methanol

ml Milliliter

mm millimeter

mM millimolar

MRT Mean residence time

N Normality

n Number of replicate

Na+ Natrium ion

NaOH Sodium hydroxide

ng Nanogram

nm nanometer

ODS Octadecylsilane

pH negative logarithm of H+ concentration

pKa Ionisation constant

xvi

PYR Pyrimethamine

r Correlation coefficient

RH Reduced haloperidol

RP Reverse phase

RP-HPLC Reversed-phase high performance liquid

chromatography

rpm revolution per minute

Rs Resolution

SD Standard deviation

Sec Second

S.E.M. Standard error of the mean

SPE Solid phase extraction

t1/2 Elimination half-life

Tmax Time to reach peak plasma conentration

USP United State Pharmacopoeia

UV Ultraviolet

v/v Volume by volume

v/w Weight by volume

Vd Volume of distribution

% Percent

± Plus/minus

α1 Adenergic receptor subtype 1

µl Microliter

µm Micrometer

< Less than

> Greater than

xvii

PERKEMBANGAN DAN PENGESAHAN KAEDAH KCPT BAGI PENENTUAN

HALOPERIDOL DAN HALOPERIDOL TERTURUN SECARA SERENTAK DI DALAM

PLASMA : APLIKASI DALAM KAJIAN FARMAKOKINETIK

Abstrak

Haloperidol merupakan suatu drug antipsikotik yang tipikal dan secara

kimianya daripada kumpulan butirofenon. Suatu kaedah kromatografi cecair

berprestasi tinggi yang peka dan selektif dengan pengesan ultra-lembayung telah

diperkembangkan bagi penentuan haloperidol dan haloperidol terturun secara serentak

di dalam plasma. Drug dikesan pada 230 nm. Pemisahan kromatografi dilakukan

dengan menggunakan turus KCPT Inertsil C8-3 (150 x 4.6 mm, 5µm). Fasa bergerak

yang digunakan terdiri daripada 50 mM larutan penimbal fosfat (pH 5.0) – metanol

(51:49, v/v) dengan kadar aliran fasa bergerak 1.0 ml/min. Pengekstrakan cecair-

cecair yang mudah telah dihasilkan dan pirimetamina digunakan sebagai piawai

dalaman. Purata peratus pengembalian bagi haloperidol, haloperidol terturun dan

pirimetamina adalah 82.4, 82.1 dan 82.0% masing-masing. Kaedah ini menunjukkan

selektiviti yang baik di mana tidak terdapat gangguan daripada puncak-puncak drug

antipsikotik yang lazim digunakan. Keluk kalibrasi adalah linear bagi julat kepekatan 1-

60 ng/ml dengan pekali korelasi ( r ) > 0.999. Peratus pekali variasi bagi kepersisian

kaedah dalam sehari dan hari ke hari adalah kurang daripada 5%. Had pengesanan

dan had kuantifikasi bawah adalah 0.5 ng/ml dan 1.0 ng/ml masing-masing bagi

haloperidol dan haloperidol terturun.

Stok piawai yang dilarutkan dalam metanol didapati stabil selama tiga

bulan pada suhu -20°C. Manakala sampel-sampel plasma didapati stabil selama 6

xviii

bulan pada suhu-20°C and -85°C Kaedah ini telah diaplikasi dengan jayanya dalam

kajian farmakokinetik haloperidol di dalam tikus di mana haloperidol diberikan secara

dos oral (2.5 mg/kg). Nilai purata t1/2, Cmax, tmax, AUC(0-∞), CL dan Vd adalah 6.2 ± 2.6

h, 24.9 ± 7.2 ng/ml, 2.0 ± 1.9 j, 214.1 ± 76.0 ng.j/ml, 213.0 ± 61.9 ml/min/kg dan 110.3

± 46.3 l/kg masing-masing. Kaedah analisis bagi haloperidol dan haloperidol terturun

yang telah dihasilkan didapati sesuai bagi kajian farmakokinetik.

xix

DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR SIMULTANEOUS

DETERMINATION OF HALOPERIDOL AND REDUCED HALOPERIDOL IN

PLASMA: APPLICATION IN PHARMACOKINETIC STUDY

Abstract

Haloperidol is a typical antipsychotic drug that chemically belongs to

butyrophenone group. A sensitive and selective reversed-phase HPLC method with

ultraviolet detection was developed for the simultaneous determination of haloperidol

and reduced haloperidol in plasma. Drugs were detected at 230 nm. The

chromatographic separation was performed on an Inertsil C8-3 (150 x 4.6mm, 5 µm)

HPLC column. Mobile phase composed of 50 mM phosphate buffer pH 5.0 and

methanol (51:49, v/v) and eluted at 1.0 ml/min. A simple liquid-liquid extraction was

carried-out using pyrimethamine as an internal standard. The mean extraction recovery

for haloperidol, reduced haloperidol and pyrimethamine were 82.4, 82.1 and 82.0%

respectively. The method showed good selectivity with respect to commonly

administered psychotropic drugs. Calibration curve was linear over the concentration

range 1 - 60 ng/ml with the correlation coefficient (r) > 0.999. The within-day and day-to

-day assay precision was less than 5%. Limit of detection and lower limit of

quantification were 0.5 ng/ml and 1 ng/ml respectively for haloperidol and reduced

haloperidol.

Stock standard of haloperidol and reduced haloperidol in methanol were

stable up to three months at -20°C. Plasma samples spiked with haloperidol and

reduced haloperidol was stable up to 6 months at -20°C and -85°C. The method was

successfully applied to pharmacokinetic study of haloperidol in rats following oral dose

of haloperidol (2.5 mg/kg). The mean t1/2, Cmax, tmax, AUC (0-∞), CL and Vd were 6.2 ± 2.6

xx

h, 24.9 ± 7.2 ng/ml, 2.0 ± 1.9 h, 214.1 ± 76.0 ng.h/ml, 213.0 ± 61.9 ml/min/kg and

110.3 ± 46.3 l/kg respectively. The analytical method for haloperidol and reduced

haloperidol assay is found to be suitable for pharmacokinetic studies.

1

CHAPTER 1 INTRODUCTION

1.1 PSYCHOSIS

Psychosis is a mental disorder which involves striking disturbances of

thought, perception, affect, and behaviour (Pantelis et al., 2003). The expression of

psychotic symptoms varies over time and across patients, however the cumulative

effects of the illness are always severe and usually long lasting (Sadock & Sadock,

2000). There are various types of psychotic disorders such as anxiety, bipolar disorder

(manic disorder), depression and schizophrenia (Grilly, 1994). These disorder a briefly

discussed in the following sections.

1.1.1 ANXIETY

Anxiety is a normal response to psychological stress induced by either

physical or perceived threat. However, a malfunctioning anxiety response could lead to

anxiety disorders. Anxiety disorders are further divided into five types; such as panic

disorder, social phobia, obsessive compulsive disorder, generalised anxiety disorder

and posttraumatic stress disorder (Leveleki et al., 2006; Ballenger, 1999). Patients

suffering from anxiety disorder are usually treated either by medication or

psychotherapy (Ballenger, 1999).

1.1.2 MANIA OR BIPOLAR DISORDER

Mania or bipolar disorder is a brain disorder that causes unusual shifts

in a person’s mood, energy and ability to function. During a manic episode, the mood

disturbances are severe enough to cause significant impairment in occupational

functioning or in otherwise normal social activities (Schapiro, 2005). Bipolar disorder is

further classified into two groups namely Bipolar I and Bipolar II. Bipolar I is

characterised by one or more manic or mixed episodes. Bipolar II is characterised by

2

recurrent episodes of major depression and hypomania. Bipolar disorder affects

approximately three to five percent of the world’s population, and affects both sexes

equally in all age group (Shastry, 2005).

1.1.3 DEPRESSION

Depression is defined as a feeling of intense sadness. Depressed

individuals tend to be obsessed with personal failings, are apathetic and socially

withdrawn (Malatynska & Knapp, 2005). Depression disorder can be transmitted in the

form of negative emotions from living or non-living beings to susceptible host.

Therefore, depression is also known as communicable disorder (Kalra, 2004).

Symptoms of depression disorder can last periods of time, sometimes several to years

(Gard, 2001).

1.1.4 SCHIZOPHRENIA

Schizophrenia is a complex cognitive disorder comprising of a variety of

alterations in attention, working memory, language, response monitoring and inhibition

(Freedman, 2003). Schizophrenia is treated as a debilitating disorder of the central

nervous system. Its symptoms are divided into two classes namely positive symptoms

and negative symptoms. Positive symptoms include hallucinations, delusions,

conceptual disorganization, where negative symptoms include social withdrawal,

blunted affect, and poverty of speech (Sawa & Snyder, 2002; Donaldson et al., 1983).

This disorder reduces the ability of the individual to interact with the social.

Schizophrenia affects about 1% of the world's population (Buchanan & Carpenter,

2000). All these types of mental disorders mentioned above are treated with

antipsychotic drug that will be described in the following section.

3

1.2 ANTIPSYCHOTIC DRUG

The term antipsychotic is applied to a group of drugs used to treat

psychosis. These classes of drugs were originally called 'neuroleptics' (from the Latin

root which mean to grasp the neuron) (Nicholas, 2004). Antipsychotic drugs are

generally divided in two types; that is typical antipsychotic drug and atypical

antipsychotic drug.

1.2.1 TYPICAL ANTIPSYCHOTIC DRUG

Typical antipsychotics are also known as major tranquilizers because of

their tranquilising and sedating effects when taken in large doses. Typical antipsychotic

drugs are effective primarily against positive symptoms of schizophrenia. Such drugs

include haloperidol (HP), chlorpromazine, perphenazine, thioridazine and

trifluoperazine are examples of typical antipsychotic drug (Fig. 1.1). Chlorpromazine

was the first typical antipsychotic drug used in 1952 to treat schizophrenic patients

(Edliner et al., 2005). Antipsychotic properties of typical antipsychotics are achieved

through the antagonistic effect on dopamine receptors.

The typical antipsychotic drugs used to treat schizophrenia are highly

effective. In particular, HP, the most widely used typical antipsychotic is very efficient in

treating the positive symptoms of schizophrenia. The most predominant among these

symptoms are dystonia, parkinsonian-like syndrome, and tardive dyskinesia

(Andreassen et al., 1996).

1.2.2 ATYPICAL ANTIPSYCHOTIC DRUG

Atypical antipsychotic drugs are used to treat schizophrenic patients

and results less extrapyramidal side effects. Compared to the older ‘typical’

antipsychotic, the atypical antipsychotic drugs are equally effective against the positive

symptoms and the negative symptoms of schizophrenia. Atypical antipsychotic drugs

4

block both dopamine and serotonin (5-hyroxytryptamine) receptors. These combined

effects on both dopamine and serotonin (5-hyroxytryptamine) receptors explain the

lower extrapyramidal side effects of atypical antipsychotic drugs. Examples of atypical

antipsychotic drugs are ariprazole, risperidone, clozapine, olanzapine, quetiapine and

ziprasidone (Fig. 1.2).

1.3 HALOPERIDOL

HP is {4-[4-(p-chlorophenyl)-4-hydroxypiperidino] 4’-flurobutyrophenone}

a typical butyrophenone antipsychotic drug. HP is a tertiary amine that occurs as a

white or almost white powder, is practically insoluble in water, and is slightly soluble in

alcohol, methanol (MeOH) and methylene chloride. The melting point of HP is 150 to

153°C. The empirical formula for HP is C21H23ClFNO3 (B P, 2003). The drug has a

pKa value of 8.3 (AHFS, 2000).

HP is effective in the treatment of many psychotic disorders such as

hyperactivity, agitation and mania (Robert & Allain, 2001). HP effectively treats positive

symptoms of schizophrenia (Glick et al., 2001) while ineffective against negative

symptoms of schizophrenia. HP is also used in the treatment of neurological disorders

such as Gilles de la Tourette syndrome, Huntington’s chorea and acute/chronic brain

syndrome (Silver et al., 2001; Barr et al., 1988; Maltbie & Cavenar, 1977). Long-term

use of HP can result in side effects resembling Parkinson’s disease and tardive

dyskinesia, an irreversible motor disorder (Fuxe et al., 1989).

Nearly 40 years after its discovery, HP is still one of the most popular

drug used for the management of various classes of psychosis. The pharmacology of

HP has been extensively reported (Janssen, 1967). Different assay methods have

been described in the literature, which will be discussed in the following section.

5

C CH2 CH2 CH2 N

OH

Cl

F

O

a. Haloperidol

S

N

(CH2)3

N

N

CF

CH3

S

N

(CH2)3

N

N

Cl

(CH2)2

OH

b. Trifluoperazine c. Perphenazine

S

N

(CH2)2

N CH3

SCH3

S

N

(CH2)3

N

H3C CH3

Cl

d. Thioridazine e. Chlorpromazine

Figure 1.1 Structural formula of typical antipsychotic drugs.

6

N

NH

N

N

CH3

Cl

N

NH

S

N

N

CH3

CH3

a. Clozapine b. Olanzapine

N N

N

S

H

H

(CH2)2OCH2CH2OH

N

N

O

CH3

N

NO

F

c. Quetiapine d. Risperidone

Figure 1.2 Structural formula of atypical antipsychotic drugs.

7

1.3.1 ASSAY OF HP AND RH IN BIOLOGICAL FLUIDS

Several analytical methods have been reported in the literature for the

determination of either HP or RH or both drugs in biological fluids. All these methods

involve various types of detection techniques (Table 1.1). Selection of a detection

technique depends on the types of analytes, and the sensitivity required. For routine

analysis, the most commonly used detection techniques are ultraviolet (UV) and

electrochemical (EC) methods.

A number of techniques are available in the literature for the

determination of HP in plasma. Some of these include gas chromatography (Bianchetti

& Morselli, 1978), radioimmunoassay (Clark et al., 1977) and UV methods (Kogan et

al., 1983; Miyazaki & Arita, 1981). Pharmacological studies showed that reduced

haloperidol (RH), metabolite of HP is pharmacologically active. Therefore, a routine

method for determination of plasma level should include both HP and RH.

Simultaneous determinations of HP and RH in human plasma by HPLC system

coupled with UV (Miller & Devane, 1986; Cahard et al., 1990; Park et al., 1991) or EC

(Aravagiri et al., 1994; Korpi et al., 1983; Eddington & Young, 1988) method have been

reported.

Although HPLC with EC detection is highly sensitive, it involves the use

of high voltage that can be problematic in monitoring HP plasma levels (Parkinson,

1985). Furthermore, increased interference from co-administered drugs is observed in

assay (Pan et al., 1998). Several other methods reported in the literature include gas

chromatography with nitrogen-phosphorus or electron-capture detector (Bianchetti &

Morselli, 1978; Franklin, 1980; Forsman et al., 1974) and radioimmunoassay (Clark et

al., 1977; Browning et al, 1985), are relatively time consuming and not a common

routine method in many therapeutic drug monitoring laboratories. The

8

radioimmunoassay method is very cumbersome and lack specificity and sensitivity and

with it, RH is determined by an indirect method.

Recently, liquid chromatography in combination with mass

spectrometric detection (Hempenius et al., 1999; Hoja et al., 1997) has been reported

for the analysis of HP. Sophisticated detector such as mass spectrometry coupled with

liquid chromatography or gas chromatography is admittedly highly sensitive. However,

it is very expensive and requires great expertise in order to operate. With such

considerations in mind, a method based on UV detection is still the most practical for

routine applications and therefore is the method of choice in this thesis.

For analysis of drugs and its metabolites in plasma, sample preparation

is very important. HP and RH were extracted from biological fluids either by liquid-liquid

extraction (LLE) or solid phase extraction (SPE) (Table 1.2). However, LLE is the most

popular and convenient method for routine analysis of HP and RH determinations

(Korpi et al., 1983; Miller & Devane, 1986; Hariharan et al., 1989; Park et al., 1991).

Only a few groups used the SPE method (Hempenius et al., 1999; Hoffman & Edkinds,

1994; Cahard et al., 1990). LLE extraction involves single, double or multiple step of

extraction. Extraction efficiency partly depends on the types of extraction solvents,

sample preparation pH, back extraction solvents and physiochemical properties.

Different types of extraction solvents are used to extract drug from plasma. In most

case mixtures of organic non-polar and polar solvents are employed. For HP and RH

determinations commonly used non-polar solvents are pentane, hexane and heptane

and polar solvents are isoamyl alcohol and isopropanol (Table 1.2). However, from the

reported recovery data presented in Table 1.2 it is difficult to conclude which

combination of extraction solvents were the best, since extraction performance depend

on several factors.

9

To keep drugs in a neutral state, basifying agents are generally added

to plasma. Sodium hydroxide (Hariharan et al., 1989; Miller & Devane, 1986; Jatlow et

al., 1982) and sodium carbonate (Aravagiri et al., 1994; Midha et al., 1988) at different

concentrations were used as basifying agents for HP determination in plasma.

Different types of solvents are used to back extract the drug from

organic phase into aqueous phase. For HP and RH determination hydrochloric acid

(Midha et al., 1988; Miller & Devane, 1986; Hoja et al., 1997), perchloric acid

(Hariharan et al., 1989) and sulfuric acid (McBurney & George, 1984; Park et al., 1991)

were used as back extraction solvents. However, organic acid (perchloric acid and

acetic acid) are the preferred back extraction solvent as compared to inorganic acid

(hydrochloric acid and sulphuric acid). A much cleaner blank chromatogram was

obtained using perchloric acid rather than hydrochloric acid or sulphuric acid

(Hariharan et al., 1989).

1.3.2 PHARMACOLOGY OF HP

A pharmacological action of drug is dependent upon its chemical

structure, any change in chemical structure may change the drug action. The exact

drug action also depends on its binding affinity to the receptor types. Drugs combine

with these receptors to produce their pharmacological effect (Gard, 2001).

Pharmacological activity of HP is related to its affinity for the dopamine

receptors. (Beuger et al., 1996; Creese et al., 1976; Seeman & Lee, 1975; Seeman et

al., 1974). Dopamine receptors are classified into five subtypes such as D1, D2, D3, D4

and D5 (Dearry et al., 1990; Grandy et al., 1991; Monsma et al., 1990; Sokoloff et al.,

1990; Sunahara et al., 1991; Vantol et al., 1991). These receptors are further divided

into two subfamilies such as the D1-like receptors (D1 and D5) and the D2-like receptors

(D2, D3 and D4)(Tarazi, 2001). The binding of HP to the postsynaptic D2-like receptors

10

is believed to mediate the therapeutic effects (Creese et al., 1976; Seeman & Lee,

1975; Seeman et al., 1974). In addition to the specific functions of several subtypes,

dopamine receptors also have distinct functions based on their location at the neuron,

i.e. pre-synaptically or post-synaptically (Nagy et al., 1978). Post-synaptic dopamine

receptors are necessary for signal initiations or transmission, whereas pre-synaptic

dopamine receptors, located on the cell bodies and axon terminals, function as

autoreceptors through which the release of dopamine can be regulated.

HP, which is a dopamine antagonist, mimics the dopamine in binding to

the dopamine receptor. There appears to be a very narrow range between the effective

therapeutic dose for the management of acute psychotic disorder and that, which

causes extrapyramidal side effects (AHFS, 2000). Therefore, the determination of

antipsychotic drugs plasma concentration in psychiatric patient is important, especially

in case of the HP, due to large variability between individuals. When HP is metabolised

in the liver it produces the metabolite RH. This reduced metabolite, RH is then oxidised

back to HP. Therefore, the influence of HP and RH on clinical responses requires the

monitoring of plasma levels for better patient management during HP therapy.

11

Table 1.1 Summary of analytical method for HP determination in biological matrix.

References Detector Internal standard Column Mobile phase Extraction method

LOQ/ LOD

Recovery (%)

Miyazaki & Arita. (1981) Jatlow et al. (1982) Kogan et al. (1983) Korpi et al. (1983) Dhar & Kutt. (1984) Miller & Devane. (1986)

HPLC-UV 250 nm HPLC-UV 195 nm HPLC-UV 254 nm HPLC-EC HPLC-UV 254 nm HPLC-UV 196 nm

Diphenylamine Desipramine Chlorohaloperidol Chlorohaloperidol Chloro-substitute analog Chlorohaloperidol

C18 Nucleosil 250 x 4 mm,

i.d. 5µm C18 Bio-sil ODS 250 mm

i.d. 10 µm C18 Absorbosphere 100 x 4.6 mm, i.d. 3 µm

µBondapak CN 300 x 3.9 mm,

i.d. 10 µm C8 bonded RP 250 x 4.6 mm,

i.d. 5 µm C18 250 x 4.6 mm,

i.d. 10 µm

Methanol-0.2 M ammonium acetate (63:37, v/v) Acetonitrile-Potassium dihydrogen phosphate pH3.8-4 (40:60, v/v) Acetonitrile-0.4 ml concentrated sulfuric acid, 1.1 g tetramethyl ammonium chloride, 0.5 ml triethylamine per liter (45:55, v/v) Acetonitrile-potassium phosphate buffer pH 6.8 (45:55, v/v) Acetonitrile-methanol-16.5mm disodium hidrogen phosphate pH5 (45:25:30, v/v/v) Acetonitrile-100 mm monobasic potassium phosphate pH 3.8-4 (55:45, v/v) flow rate : 2.0 ml/min

LLE LLE LLE LLE LLE LLE

5 ng/ml (LOD) 1 ng/ml (LOD) 2 ng/ml (LOD) 0.5 ng/ml (LOD) 1 µg/l (LOD) 2 ng/ml (LOD)

HP :102% HP :78% HP :80% HP:92% RH:93% HP:89% HP:86% RH:92%

12

Table 1.1 continued

References Detector Internal standard Column Mobile phase Extraction method

LOQ/ LOD Recovery (%)

Eddington & Young. (1988) Hariharan et al. (1989) Cahard et al. (1990) Wilhelm & Kemper. (1990) Park et al. (1991) Aravagiri et al. (1994)

HPLC-EC HPLC-coulometri HPLC-UV 220 nm Photo-diode HPLC-UV 214 nm HPLC-EC

Chlorohaloperidol Bromoperidol Chlorohaloperidol Clonazepam Bromoperidol Chlorohaloperidol

Nitrile bonded LC-PCN 150 x 4.6 mm,

i.d. 5 µm C8 RP (DB) 250 x 4.6 mm,

i.d. 5 µm

C18 µBondapak 300 x 3.9 mm,

i.d. 10 µm Silica 100 RP-18 120 x 4 mm,

i.d. 5 µm C18- Nova Pak 150 x 2.9 mm,

i.d. 4 µm Ultrasphere Cyano 250 x 4.6 mm,

i.d. 5 µm

Acetonitrile-1-propanol-10 mm dipotassium hidrogen phosphate pH 6.8 (30 :20 :50, v/v/v) flow rate : 1.1ml/min Acetonitrile-methanol-50 mM KH2PO4 pH 7 (20:40:40, v/v/v) Acetonitrile-water-0.025 M potassium dihydrogenphosphate (45:5:50, v/v/v) flow rate 0.8 ml/min Gradient : ethanol-acetonitrile-0.05 M phosphate buffer pH2.9 (22:8:70, v/v/v) Acetonitrile-methanol-0.5 M potassium dihydrogen phosphate (31:11:58, v/v/v/) Flow rate : 0.6 ml/min Acetonitrile-methanol-0.04M ammonium acetate pH 6.8 (86:6:8, v/v/v)

SPE LLE SPE SPE LLE LLE

20 pg (LOD) 50 ng/l (LOD) 1 ng/ml (LOQ) 450 pg/ml (LOQ) 0.5 ng/ml (LOD) 0.1 ng/ml (LOQ)

HP :93% RH :90% HP :83% RH :76% HP :101% HP :95% RH :73% HP :68% RH:86%

13

Table 1.1 continued

References Detector Internal standard Column Mobile phase Extraction method

LOQ/ LOD Recovery (%)

Hoffman & Edkins. (1994) Hoja et al. (!997) Pan et al. (1998) Hempenius et al. (1999) Titier et al. (2003)

HPLC-coulometri LC-EC-MS HPLC-UV 220 nm HPLC-EC LC-MS HPLC-UV 240 nm

Chlorohaloperidol Chlorohaloperidol Chlorohaloperidol Haloperidol D4 Methylrisperidone

Microsorb – 100 x 4.6 mm,

i.d. 3 µm C18 Nucleosil 150 x 1 mm,

i.d. 5 µm C18 Symmetry 150 x 3.9 mm, i.d. 5 Altima CN 250 x 4.6 mm,

i.d. 5 µm C18 Symmetry 100 x 4.6 mm,

i.d. 3.5 µm C8 Symmetry i.d. 250 x 4.6 mm,

Methanol-50mM sodium phosphate pH 6.5 (50:50, v/v) Flow rate : 0.5ml/min Acetonitrile-0.04M ammonium formate (45:55, v/v) Acetonitrile-0.02M sodium dihydrogen phosphate pH 3.8 (29:71, v/v) Acetonitrile-Methanol-0.04M sodium dihydrogen phosphate pH 6.8 (35:20:45, v/v/v)l Methanol-0.2% formic acid (50:50, v/v) Gradient : Acetonitrile-50mM NaH2PO4

SPE LLE LLE SPE LLE

0.5 ng/ml (LOD) 0.075 ng/ml (LOD) 2 ng/ml (LOQ) 0.5 ng/ml (LOQ) 0.1 (LLQ) 5 ng/ml (LOQ)

HP :80% HP :58% RH:70% HP :99-104% HP :83-121% HP:69%

Abbreviation: HPLC-UV = High performance liquid chromatography-Ultraviolet detector; HPLC-EC= High performance liquid chromatography-electrochemical detector; LC-EC-MS= liquid chromatographic-electrospray mass spectrometric; LC-MS= liquid chromatographic mass spectrometric; i.d.= internal diamension; mm=milimeter; µm=mikrometer; nm=nanometer; LLE = liquid-liquid extraction; SPE= solid phase extraction; LLOQ=lower limit of quantification, LOQ=limit of quantification; LOD=limit of detection; HP=haloperidol; RH=reduced haloperidol.

14

Table 1.2 Summary of extraction method for HP determination in biological matrix. References Extraction solvent Basifying agent Back extraction solvent Reconstitute solvent

Titier et al. (2003) Hexane : isoamylalcohol (99:1, v/v) (7 ml)

Sodium hydroxide (2 N, 100 µl))

Hydrochloric acid(0.05 M, 200 µl)

Direct injection

Pan et al. (1998) Hexane : isoamylalcohol (100:2, v/v) (5 ml)

Sodium hydroxide (2 N, 200µl)

Hydrochloric acid (0.1 N, 150 µl)

Mobile phase

Hoja et al. (1997) Hexane : isoamylalcohol (99:1, v/v) (8 ml)

Sodium hydroxide ( 2M, 500µl)

Hydrochloric acid (0.2 M, 1ml)

Mobile phase (50 µl)

Aravagiri et al. (1994)

Pentane:methylchloride (90:10, v/v) (7 ml)

Saturated sodium carbonate (0.5 ml)

Acetonitrile (150µl)

Park et al. (1991) Hexane : isoamylalcohol ( 98 :2, v/v) (5 ml)

Sodium hydroxide (2M, 300-500 µl )

Sulphuric acid (0.025 M, 120 µl )

Direct injection

Hariharan et al. (1989)

Pentane : isopropanol (95:5, v/v) (6 ml)

Sodium hydroxide (14.8 mol/l, 5 drop)

Perchloric acid (100 mM, 2ml)

Mobile phase (100µ)

Midha et al. (1988) Isopropanol : n pentane ( 5 :95, v/v) (10 ml)

Saturated sodium carbonate (0.5ml)

Hydrochloric acid (0.1 N, 1ml)

Acetonitrile (40µ)

15

Table 1.2 continued

References Extraction solvent Basifying agent Back extraction solvent Reconstitute solvent

Miller & Devane. (1986)

Hexane : isoamylalcohol (98 :2, v/v) (6 ml)

Sodium hydroxide (5 M, 100 µl )

Hydrochloric acid (0.1 M, 100µl)

Direct inject

McBurney & George. (1984)

Heptane : isoamylalcohol (98.5 :1.5, v/v)

Sodium hydroxide (2 M, 1 ml)

Sulphuric acid (0.005 M, 2ml)

Methanol

Dhar & Kutt. (1984) Heptane : isoamylalcohol (15% in 1L) (5 ml)

Sodium hydroxide (1 mol/l, 0.5 ml)

Sulphuric acid (5 mM, 0.6ml)

Direct injection

Jatlow et al. (1983) Hexane : isoamylalcohol ( 98 :2, v/v) (10 ml)

Sodium hydroxide (2 M, 0.2 ml)

Hydrochloric acid (0.1 M, 150µl)

Direct injection

Miyazaki & Arita. (1981)

Diethylether (4 ml) Sodium hydroxide (1 N, 1 ml)

Hydrochloric acid (0.1 N, 3.5ml)

Direct injection

Bianchetti & Morselli. (1978)

Diethyleter (5 ml) Sodium hydroxide (2 N, 200 µl )

Hydrochloric acid (0.2 N, 2.5ml)

Hempenius et al. (1999)

Solid phase extraction

Hoffman & Edkinds. (1994)

Solid phase extraction

Cahard et al. (1990) Solid phase extraction

Abbreviation: ml= mililiter; µl=microliter; M=molarity; N=normality; l=liter; mol/l=mol per liter; v/v=volume by volume; mM=milimolar

16

1.3.3 PHARMACOKINETICS OF HP AND RH

Numerous articles related to the absorption, distribution, metabolism

and excretion of HP in animal (Braun et al., 1967; Soujin et al., 1967) and human

(Cressman et al., 1974) have been reported. In most of these studies, pharmacokinetic

characteristics of HP are usually reported after a single oral and intramuscular (IM)

(Cressman et al., 1974) or intravenous (IV) administration in healthy volunteers (Holley

et al., 1983; Chakraborty et al., 1989) and in schizophrenic patients (Cheng et al.,

1987). A summary of the HP and RH pharmacokinetic parameters in animal and

human are shown in Table 1.3.

HP is a lipophillic compound and therefore is rapidly absorbed from the

gastrointestinal tract following oral administration, and undergoes a first pass

metabolism in the liver. Due to the first-pass metabolism in the liver, plasma levels of

HP occurs within 2 to 6 h after oral dosing (Kunka & Perel, 1989) and about 20 min

after IM administration (Cressman et al., 1974) and the systemic bioavailability is

approximately 75% (Adam & Fernandez 1987; Javaid, 1994). Oral bioavailability is

about 65% after first pass metabolism (Holley et al., 1983).

HP is bound to plasma protein and the free fraction in serum is reported

to be about 8 %( Forsman & Ohman, 1977a). It is widely distributed in the body and

crosses the blood brain barrier and is also distributed in breast milk.

Pharmacokinetic studies of RH in normal volunteers (Chakraborthy et

al., 1989; Midha et al., 1989) and schizophrenic patients (Chang et al., 1989a) have

been reported. In a study by Chang et al (1989a), a single 10 mg dose of reduced

haloperidol was given orally to seven male schizophrenic patients (age 43.4± 8.6

years; weight 54.6 ± 9.0 kg). The pharmacokinetic parameter of RH is shown in Table

1.3.

17

Table 1.3 Summary of pharmacokinetic for HP and RH Model Route No Dose

(mg) t1/2 (h) Vd (l/kg) CL (l/h)

AUC (ng.h/ml) References

Haloperidol

Cheng et al. (1987)

Human (67 ± 16kg)

Intravenous Oral

6 8

1.5 - 5.0 2.0 - 5.0

18.8 ± 4.7 18.1 ± 4.5

9.5 ± 1.9

33 ± 7.8 NR

Holley et al. (1983)

Human (70.9 ± 7.0kg)

Intravenous Oral

6 8

0.125 0.503

26.2 ± 8.0 14.5 ± 3.2

21.7 ± 6.9

49.2 ± 12 NR

Magliozzi & Hollister. (1985)

Human Intravenous Oral

6 6

0.125 0.500

15.1 ± 2.5 17.5 ± 8.7

NR NR NR

Forsman et al. (1974)

Human Intravenous 3 5 12.6 27 NR NR

Cheng & Paalzow. (1992)

Rats (190 - 250g)

Intravenous 6 - 8 0.5 1.0 2.5

1.53 1.54 1.54

4.3 5.6 6.5

86.2a

82.3a

80.7a

5724.5 ± 4.6 11874.2 ± 3.0 32110.4 ± 2.9

Wurzburger et al. (1981)

Rats (300 - 350g)

Intravenous 0.25 0.5 1.0

2.69 2.60 2.50

3.33 4.60 4.50

53.2b

46.7b

42.2b

80.1 174 378

Reduced haloperidol

Chang et al. (1989a)

Human (54.6 ± 9.0 kg)

Oral 7 10 67.0 ± 51.3 46.0 ± 20.2 0.60 ±0.30c 448.5 ± 336.0

d

Note: a = Clearance reported at unit ml min-1 kg-1 ; b = Clearance reported at unit ml/min c = Clearance reported at unit l/h/kg and d = AUC reported at unit µg.l/h Abbreviations: t1/2 = elimination half-life; Vd=apparent volume of distribution; CL=clearance; NR=not reported

18

Table 1.4 Concentration of HP and RH in rat tissues after a single intraperitoneally injection of HP or RH ( 1mg/kg body weight) ( Korpi & Wyatt, 1984).

Tissue/time after injection HP treatment ______ RH treatment ____________ HP RH HP RH ________________________________________________________________________________________________________________ Plasma 10 min 0.2 ± 0.1 n.d. 0.0 ± 0.0 0.1 ± 0.1 30 min 0.1 ± 0.0 n.d. 0.0 ± 0.0 0.1 ± 0.0 120 min 0.1 ± 0.0 n.d. 0.0 ± 0.0 0.0 ± 0.0 Liver 10 min 24.0 ± 7.8 0.4 ± 0.2 6.8 ± 0.5 1.8 ± 0.1 30 min 6.2 ± 0.2 0.1 ± 0.0 3.1 ± 0.1 0.8 ± 0.1 120 min 2.9 ± 0.2 0.0 ± 0.0 2.1 ± 0.1 0.3 ± 0.0 Corpus striatum 10 min 3.4 ± 1.3 n.d. 0.2 ± 0.0 1.6 ± 1.3 30 min 3.2 ± 0.5 n.d. 0.5 ± 0.1 2.3 ± 0.9 120 min 1.2 ± 0.2 n.d. 0.6 ± 0.1 0.6 ± 0.2

Drug concentrations are given as mean ± S.E.M. (n=4) in µmol/l for plasma and in µmol/kg wet weight for liver and corpus striatum. n.d.= not detected.

19

Table 1.5 Concentration of HP and RH in striatum and plasma after repeated IP injections of HP(Chang et al., 1988).

Dose Drug concentration_____________________________________________________________________________________ (mg/kg) Striatum (ng/g) _________________________ Plasma (ng/ml)___________________________________ HP RH HP RH

0.1 138 ± 5 168 ± 10 1.8 ± 0.2 5.0 ± 0.4 0.5 220 ± 11 433 ± 16 4.4 ± 0.4 28.8 ± 2.0 2 436 ± 17 2158 ± 155 11.1 ± 0.6 149.6 ± 6.0

All values are mean ± S.E.M. of six to eight animals Table 1.6 Pharmacokinetic parameters after administration of HP and RH.

References Subject No Drug Dose pharmacokinetic parameters_________________________ AUC (ng.h/ml) CL (l/kg) t1/2 (h)

Chakraborty Healthy volunteers 15 HP (given HP 5 mg) 70.7 ± 33.4 NR 53.9 ± 24.5 et al., 1989 77.6 ± 10kg RH (given HP 5 mg) 62.0 ± 103.1 NR 69.2 ± 23.9 HP (given RH 5 mg) a NR a RH (given RH 5 mg) 54.5 ± 20.9 NR 73.2 ± 29.4

Jann et al., Schizophrenic 6 HP (given HP 10 mg) 200.5 ± 99.2b 1.1 ± 0.3c 21.5 ± 9.1 1990 43 - 66 kg RH (given HP 10 mg) 85.0 ± 72.2b 6.2 ± 8.8c 34.0 ± 22.5 HP (given RH 10 mg) 48.2 ± 66.4b 11.5 ± 10.9c 36.7 ± 48.0 RH (given RH 10 mg) 432.5 ± 336.3b 0.7 ± 0.6c 71.9 ± 61.2

Note: a= could not be calculated in 14 out of 15 volunteers because HP not determined given RH dose. b= AUC reported at unit mcg.h/l. CL = Clearance reported as unit l/h/kg. Abbreviation : AUC = Area under the curve; HP = Haloperidol; RH = Reduced haloperidol; kg=kilogram; mg=miligram.

20

The liver is the major site of HP metabolism (Korpi et al., 1985). The

metabolism of the HP in humans involves the initial cleavage of the molecule at the C-

N bond of the central chain to form an inactive piperidine and 4-fluorobenzoylpropionic

acid metabolites (Forsman et al., 1977) (Figure 1.3) and the formation of a conjugate

with glucuronic acid at the hydroxy group (Oida et al., 1989). HP is also metabolised

through reduction at the benzylic ketone group to form an alcohol metabolite, known as

reduced haloperidol (RH) (Forsman & Larsson, 1978).

HP is excreted slowly in the urine and faeces. About 30% of a given

dose is excreted in urine and about 20% of a given dose in faeces via biliary

elimination (Beresford & Ward, 1987). Only 1% of a given dose is excreted as

unchanged drug in the urine (Forsman et al., 1977). There is also evidence of

enterohepatic recycling (Chakraborty et al., 1989).

The interconversion between HP and RH are species specific. RH is

oxidised back to HP in rats (Table 1.4) (Korpi & Wyatt 1984; Korpi et al., 1985a), while

in guinea pigs reversible metabolism of HP and RH (reduction and oxidation of both

compounds) were found (Table 1.5) (Korpi et al., 1985, 1985a; Chang et al., 1988,

1991). The presence of RH in the plasma patients treated with HP was first reported by

Forsman & Larsson (1978). However, the back conversion from RH to HP in human

was not elucidated until 1987. Back conversion of HP to RH in humans was detected in

plasma from healthy volunteer subjects. Healthy volunteer subjects were

administration of a single oral 5 mg dose of RH (Midha et al., 1987). Two years later,

these authors reported the interconversion between HP and RH in 14 of 15 normal

volunteers who received a single 5 mg dose of HP and RH separately (Chakraborty et

al., 1989; Midha et al., 1989). Further, Chang et al. (1989a) also reported the reversible

metabolism of HP and RH in schizophrenic patients (Table 1.6). The enzyme

21

responsible to produce RH from HP is characteristic of a ketone reductase that is

present in human and guinea pig hepatic cytosol (Inaba & Kovac, 1989). The reductase

activity was dependent on nicotinamide-adenine-dinucleotide-phosphate as a cofactor

for both humans and guinea pigs.

Several metabolic pathways involved in the metabolism of HP and RH

are mediated by cytochrome P450 isoenzymes (CYP) (Usuki et al., 1998). CYP3A4

plays an important role and is responsible for the N-dealkylation of HP and RH (Pan et

al., 1997; Pan et al., 1998a; Fang et al., 1997), the back oxidation of HP to RH (Pan et

al., 1998a, Kudo & Odomi, 1998; Avent & Gillam, 1998; Fang et al., 1997) and the

formation of pyridinum metabolite (Avent & Gillam, 1998; Fang et al., 1997; Eyles et al.,

1996). There are however, in the literature, several suggestions based on in vivo and in

vitro studies, that CYP2D6 (Inaba et al., 1985; Tyndale et al., 1991) could also be

involved, but the metabolic pathways are not known.

22

C

OH

CH2 CH2 CH2 N

OH

Cl

F

C CH2 CH2 CH2 N

OH

Cl

F

O

C CH2 CH2 CO2HF

O

N

OH

Cl

4-Fluorobenzoylpropionic acid Piperidine metabolite

Haloperidol

Reduced haloperidol

Figure 1.3 Metabolism of HP (Forsman et al., 1977).

23

1.3.4 DRUG INTERACTIONS

A drug interaction usually refers to the modifications of the expected

drug response due to exposure of the patients to other foods or drugs administered

concomitantly. Drug interaction may include drug-drug interaction or drug-food

interaction (Shargel & Yu, 1999). Food-drug interactions can produce negative effects

in the safety and efficacy of drug therapy and in the nutritional status of the patients

(Miguel et al., 2005). Many nutrients substantially interfere with the absorption or

metabolism of drugs in the body (Anderson, 1998; Kirk, 1995). For examples, grapes

fruit juice increase the plasma level of many drugs such as lovastatin and simvastatin

due to naringin that inhibit their metabolism (Kantola et al., 1998). Fluoroquinolones

binds with iron or calcium enriched foods and antacids if administered simultaneously.

The resulting compounds excreted with little or no systemic absorption. Significant

interaction of P-glycoproteins with cyclosporine and reduce the absorption of

levofloxacin has been reported (Wallace et al., 2003).

Many drug-drug interactions are metabolism based and related to

cytochrome P450 (CYP) enzymes. The CYP enzyme system is a very large group of

enzymes encoded by the P450 superfamily. CYPs are membrane bound proteins with

an approximate molecular weight of 50 kD and contain a heme moiety (Gunaratna,

2000). Because of the diversity of the cytochrome family a nomenclature system based

on sequence identity is developed. This nomenclature is comprised of family and sub-

family systems. Family includes CYPs and is designated by a number after CYP. Sub-

family is the CYPs within the family and is designated by a letter following the number.

For example, CYP2D6 is a cytochrome P450 enzyme. It belongs to family 2 and sub-

family D. The last number 6 refers to the sequence of discovery. There are about 30

human cytochrome P450 enzymes. Only 6 of them such as CYP1A2, CYP2C9,

CYP2C19, CYP2D6, CYP2E1 and CYP3A4 are mainly involved in drug metabolism.

24

Among these CYP3A4 is the most abundant and most clinically important isoenzymes

in humans (Gunaratna, 2000).

The enzymes involved in the metabolism of drug are altered by food and

the co-administered drug. Enzyme induction is a drug or chemical-stimulated increase

in enzyme activity usually due to an increase in the amount of enzyme present.

Enzyme inhibition is substrate competition or due to direct inhibition of the drug

metabolising enzymes, mainly CYP enzymes (Shargel & Yu, 1999). The CYP enzyme

is important to the pharmacokinetics of psychotropic drugs. The result of inhibition is a

higher plasma level of drug that can cause adverse effect. The result of induction is a

lower plasma level of drug that can affect the therapeutic efficacy (Sharif, 2003).

Concomitantly administered drugs may affect the pharmacokinetics of

HP by influencing its metabolic clearance and its ability to bind to plasma proteins, and

by causing alterations in hepatic blood flow. HP appears to be a moderately extracted

drug. On a theoretical basis, alterations in protein binding of drugs with a low extraction

ratio would have no influence on the pharmacokinetics of unbound drug at steady state.

On the other hand, alterations of plasma protein binding of high extraction drugs would

be predicted to cause a change in the clearance of unbound drug (Wilkinson & Shand,

1975; Nies et al., 1976; Blaschke, 1977).

Table 1.7 shows pharmacokinetic interactions between HP and other

drugs. Anticonvulsant drugs such as carbamazepine, phenytoin and phenobarbitone

are classified as enzyme inducers. Clinically significant decrease in plasma

concentration of HP was reported when coadministered with carbamazepine (Jann et

al., 1985; Kidron, et al., 1985; Arana et al., 1986). Another similar study showed

decreased plasma concentration of HP when co-administered with phenytoin and

phenobarbitat (Linnoila et al., 1980). There have been several reports on